DNA repair pathways maintain the integrity of the genome and thereby help prevent the onset of cancer, disease and aging phenotypes. Further, many cancer treatments function by inducing genomic DNA damage. As such, the critical requirement for DNA repair proteins and pathways in response to radiation and genotoxic chemotherapeutics implicates DNA repair proteins as prime targets for improving response to currently available anti-cancer regimens. Essential to the development of specific DNA repair inhibitors is the availability of robust, highly sensitive assays to measure DNA repair capacity. In addition, defects in critical DNA repair pathways or proteins can predispose to cancer onset and may also provide an option for therapeutic selectivity. Many of these defects in the 150 or more DNA repair proteins can be detected using current omics technologies. However, there are many defects that can only be detected using functional assays such as those described herein. To effectively develop these tools, we suggest two specific aims: Aim 1 will utilize a Reverse Engineering strategy as the basis for a novel discovery platform yielding the optimal dsDNA sequence for any DNA repair or DNA binding protein. This approach will allow DNA sequence dependence of individual DNA repair enzymes to influence probe (Molecular Beacon) optimization. Aim 2 will exploit the optimized consensus sequence defined by the reverse engineering platform in Aim 1 for the development of highly selective and specific molecular beacon probes. To provide multiplexing capacity, we will optimize for multiple sets of fluor/quencher pairs and will evaluate each assay for use in 96-, 384- and 1536-well platforms to demonstrate high-throughput application. In Aim 2, we will develop and optimize three user ready real-time fluorescence-based assays (DNA Repair Lights, DNA Repair PureLights and DNA Repair CaptureLights) amenable to DNA Repair quantification using purified proteins or cell and tissue lysates. The quantitative assays proposed here will provide a rapid, high-throughput method for the discovery and validation of DNA repair inhibitors and will be a valuable platform for functional DNA Repair measurements and biomarker analysis of cell/tumor lysates or tissue aspirates. We envision this as first in a series of assays towards the development of a complete DNA Repairomics platform.